Opine dehydrogenase

 

N-(1-D-carboxylethyl)-L-norvaline dehydrogenase is part of the (D,L) stereochemistry family of opine dehydrogenases and is able to catalyse the reaction of pyruvate with hydrophobic L amino acids to form opines through reductive condensation using NAD(P)H as a cofactor. Of particular interest is the stereochemical specificity of this enzyme as its reaction is a key step in a number of synthetic routes for pharmaceuticals. The enzyme displays some sequence homology with (L,L) opine dehydrogenases and both families display homology with 2-hydroxy acid dehydrogenases.

 

Reference Protein and Structure

Sequence
Q44297 UniProt (1.5.1.28) IPR003421 (Sequence Homologues) (PDB Homologues)
Biological species
Arthrobacter sp. 1C (Bacteria) Uniprot
PDB
1bg6 - CRYSTAL STRUCTURE OF THE N-(1-D-CARBOXYLETHYL)-L-NORVALINE DEHYDROGENASE FROM ARTHROBACTER SP. STRAIN 1C (1.8 Å) PDBe PDBsum 1bg6
Catalytic CATH Domains
1.10.1040.10 CATHdb (see all for 1bg6)
Click To Show Structure

Enzyme Reaction (EC:1.5.1.28)

L-2-aminopentanoic acid zwitterion
CHEBI:58441ChEBI
+
pyruvate
CHEBI:15361ChEBI
+
hydron
CHEBI:15378ChEBI
+
NADH(2-)
CHEBI:57945ChEBI
→
(2S)-2-{[(1R)-1-carboxylatoethyl]azaniumyl}pentanoate
CHEBI:58799ChEBI
+
NAD(1-)
CHEBI:57540ChEBI
+
water
CHEBI:15377ChEBI
Alternative enzyme names: (2S)-2-((1-(R)-carboxyethyl)amino)pentanoate dehydrogenase (NAD, L-aminopentanoate-forming),

Enzyme Mechanism

Introduction

The mechanism proposal is based on the similarity in the chemistry of the opine dehydrogenase family to that of the amino acid dehydrogenases. The reaction is believed to proceed via nucleophilic attack by the L amino acid on the pyruvate with a histidine residue acting as a general acid, allowing the formation of a carbinolamine intermediate. Dehydration of this intermediate, again facilitated by acid-base action of Histidine, results in an imine intermediate which is then reduced by hydride transfer from NAD(P)H to give the product.

Catalytic Residues Roles

UniProt PDB* (1bg6)
Asp297 Asp297A Acts to alter the pKa of His 202 to allow it to function as a general acid-base catalyst at physiological pH and thus catalyse the reaction efficiently. Thus is part of an Asp-His proton relay system. electrostatic stabiliser
His202 His202A Protonates the carbonyl oxygen resulting in a carbinolamine intermediate which proceeds via hydride transfer from the cofactor to form the product. proton acceptor, proton donor
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, bimolecular nucleophilic addition, overall reactant used, intermediate formation, dehydration, aromatic unimolecular elimination by the conjugate base, hydride transfer, overall product formed, intermediate collapse

References

  1. Britton KL et al. (1998), Nat Struct Biol, 5, 593-601. Crystal structure and active site location of N-(1-D-carboxylethyl)-L-norvaline dehydrogenase. DOI:10.1038/854. PMID:9665174.

Step 1. Catalysis begins with nucleophilic attack of the amino group of L-2-aminopentanoic acid on the carbonyl of pyruvate, forming a carbinolamine intermediate. His202 acts as an acid and donates a proton to the developing alkoxide on pyruvate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp297A electrostatic stabiliser
His202A proton donor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, overall reactant used, intermediate formation

Step 2. His202 acts as a base to deprotonate the amine group of the intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp297A electrostatic stabiliser
His202A proton acceptor

Chemical Components

proton transfer

Step 3. The hydroxyl group accepts a proton from His202 and the nitrogen lone pair facilitates dehydration of the intermediate, forming and imine intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His202A proton donor

Chemical Components

dehydration, proton transfer

Step 4. Hydride transfer from NADH to the imine intermediate occurs, forming the product and NAD.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles

Chemical Components

ingold: aromatic unimolecular elimination by the conjugate base, hydride transfer, overall product formed, intermediate collapse
Download: Image, Marvin File

Step 1. Catalysis begins with nucleophilic attack of the amino group of L-2-aminopentanoic acid on the carbonyl of pyruvate, forming a carbinolamine intermediate. His202 acts as an acid and donates a proton to the developing alkoxide on pyruvate.

Step 2. His202 acts as a base to deprotonate the amine group of the intermediate.

Step 3. The hydroxyl group accepts a proton from His202 and the nitrogen lone pair facilitates dehydration of the intermediate, forming and imine intermediate.

Step 4. Hydride transfer from NADH to the imine intermediate occurs, forming the product and NAD.

Products.

Contributors

Peter Sarkies, Gemma L. Holliday, Amelia Brasnett